Protein KinaseCδ-Calmodulin Crosstalk Regulates Epidermal Growth Factor Receptor Exit from Early Endosomes

Abstract

We have recently shown that calmodulin antagonist W13 interferes with the trafficking of the epidermal growth factor receptor (EGFR) and regulates the mitogen-activated protein kinase (MAPK) signaling pathway. In the present study, we demonstrate that in cells in which calmodulin is inhibited, protein kinase C (PKC) inhibitors rapidly restore EGFR and transferrin trafficking through the recycling compartment, although onward transport to the degradative pathway remains arrested. Analysis of PKC isoforms reveals that inhibition of PKC␦ with rottlerin or its down-modulation by using small interfering RNA is specifically responsible for the release of the W13 blockage of EGFR trafficking from early endosomes. The use of the inhibitor Gö 6976, specific for conventional PKCs (␣, ␤, and ␥), or expression of dominant-negative forms of PKC, , or ⑀ did not restore the effects of W13. Furthermore, in cells treated with W13 and rottlerin, we observed a recovery of brefeldin A tubulation, as well as transport of dextran-fluorescein isothiocyanate toward the late endocytic compartment. These results demonstrate a specific interplay between calmodulin and PKC␦ in the regulation of the morphology of and trafficking from the early endocytic compartment. INTRODUCTIONThe early endosome is a highly complex and dynamic intracellular compartment involved in the sorting of endocytosed receptors and ligands, for receptor recycling or targeting to lysosomes; in addition, it participates in endosome-endosome fusion and fission events (Gruenberg, 2001). The identification of microdomains in early endosomes, together with specific molecular activities (i.e., phosphorylation of signaling proteins or ubiquitylation of receptors), suggests that sorting and exit (budding) from this compartment are finely regulated and further indicates that our knowledge of its molecular machinery is incomplete. Thus, in addition of proteins that might be involved in the formation of specific domains (domain organizers) such as Rab5, Rab4, or annexin 2, other components are also likely to be important for the integrated function of endosomal sorting and trafficking.In a previous study, we demonstrated the importance of calmodulin in the regulation of early endocytic compartment morphology as well as in the trafficking and signaling of the epidermal growth factor receptor (EGFR) in this structure (Tebar et al., 2002). Now, we analyze the molecular mechanisms and the function of calmodulin in EGFR exit from early endosomes.Calmodulin specifically interacts with the EGFR in a calcium-dependent manner (Martin-Nieto and Villalobo, 1998;Li et al., 2004). Mutations in the juxtamembrane domain (calmodulin-binding site) of the EGFR, or deletion of the basic segment (645-660 amino acids), inhibit this interaction; interestingly, this region contains the threonine 654, the target of protein kinase C (PKC). Calmodulin binding inhibits EGFR tyrosine kinase activity in vitro (San José et al., 1992) and PKC-induced phosphorylation at Thr654 (Lund et al., 1990;Bao et al., 20...

Protein Kinase Cδ and Calmodulin Regulate Epidermal Growth Factor Receptor Recycling from Early Endosomes through Arp2/3 Complex and Cortactin

“…This blockage was associated with a dramatic alteration of the morphology and size of early endosomes (Tebar et al, 2002;Lladó et al, 2004). Furthermore we showed that a cross-talk between CaM and protein kinase C␦ (PKC␦) is involved in the regulation of the budding from the early endocytic compartment (Lladó et al, 2004).Several lines of evidence implicate the actin cytoskeleton in the CaM-and PKC␦-mediated regulation of vesicular trafficking. Because actin contributes to the maintenance of organelle stability (Apodaca, 2001), swelling and morphological changes observed in response to CaM/PKC␦ activity (Lladó et al, 2004) may involve altered actin dynamics.…”

“…In particular, in the presence of W13, a highly specific CaM antagonist, trafficking is blocked in early endosomes, thereby interfering with transport toward degradation and recycling pathways. This blockage was associated with a dramatic alteration of the morphology and size of early endosomes (Tebar et al, 2002;Lladó et al, 2004). Furthermore we showed that a cross-talk between CaM and protein kinase C␦ (PKC␦) is involved in the regulation of the budding from the early endocytic compartment (Lladó et al, 2004).…”

“…In addition, cytochalasin D or YFP-cofilinS3A expression (data not shown) significantly restored EGF receptor recycling, but not its degradation, when CaM is inhibited (Supplemental Table S1). These results indicated that actin polymerization participates in the regulation of recycling as well as degradation of EGFR.We have previously shown that when CaM was inhibited, active PKC␦ was necessary to inhibit EGFR recycling from the early endocytic compartment (Lladó et al, 2004). Thus, to determine whether PKC␦ was also involved in the formation of the F-actin coat when cells were depleted of CaM, NRK cells were incubated with the PKC␦ inhibitor rottlerin.…”

“…Furthermore we showed that a cross-talk between CaM and protein kinase C␦ (PKC␦) is involved in the regulation of the budding from the early endocytic compartment (Lladó et al, 2004).…”

“…W13 treatment produced large aberrant endosomes containing early endocytic markers and internalized EGF, dextran, and transferrin (Apodaca et al, 1994;Tebar et al, 2002). The blockage in the ongoing trafficking from these early endosomes was PKC␦ dependent (Lladó et al, 2004). …”